Drosophila Argonaute2 (AGO2) has been shown to regulate expression of certain loci in an RNA interference (RNAi)-independent manner, but its genome-wide function on chromatin remains unknown. Here, we identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs). Furthermore, we identified a somatic target of AGO2 transcriptional repression, no hitter (nht), which is immersed in a LAD located within a repressive topologically-associated domain (TAD). Null mutation but not catalytic inactivation of AGO2 leads to ectopic expression of nht and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the nht TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, our findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression.

Efferocytosis triggers cellular reprogramming, including the induction of mRNA transcripts which encode anti-inflammatory cytokines that promote inflammation resolution. Our current understanding of this transcriptional response is largely informed from analysis of bulk phagocyte populations; however, this precludes the resolution of heterogeneity between individual macrophages and macrophage subsets. Moreover, phagocytes may contain so called "passenger" transcripts that originate from engulfed apoptotic bodies, thus obscuring the true transcriptional reprogramming of the phagocyte. To define the transcriptional diversity during efferocytosis, we utilized single-cell mRNA sequencing after co-cultivating macrophages with apoptotic cells. Importantly, transcriptomic analyses were performed after validating the disappearance of apoptotic cell-derived RNA sequences. Our findings reveal new heterogeneity of the efferocytic response at a single-cell resolution, particularly evident between F4/80 + MHCII Lo and F4/80 − MHCII HI macrophage sub-populations. After exposure to apoptotic cells, the F4/80 + MHCII Lo subset significantly induced pathways associated with tissue and cellular homeostasis, while the F4/80 − MHCII HI subset downregulated these putative signaling axes. Ablation of a canonical efferocytosis receptor, MerTK, blunted efferocytic signatures and led to the escalation of cell death-associated transcriptional signatures in F4/80 + MHCII Lo macrophages. Taken together, our results newly elucidate the heterogenous transcriptional response of single-cell peritoneal macrophages after exposure to apoptotic cells. The clearance of apoptotic cells, termed efferocytosis 1 , is executed billions of times each day by a diverse spectrum of macrophage (MΦ) subsets in humans 2. This process induces an active mRNA transcriptional response within the phagocyte that is necessary to maintain tissue homeostasis and a nonphlogistic milieu 3,4. Additionally, efferocytosis within an injured tissue promotes the secretion of anti-inflammatory cytokines 5 and pro-resolving mediators 6 contributing to protective responses during tissue repair 7. However, impaired efferocytosis contributes to failed inflammation resolution and therefore is a significant feature of chronic inflammation diseases 8. For instance, in advanced atherosclerosis defective efferocytosis induces plaque necrosis and inflammation leading to subsequent plaque disruption and thrombosis 9,10. Genetic fate-mapping combined with parabiosis and adoptive transfer approaches have revealed that macrophage diversity may originate from two broad ontogenetic categories 11,12. This includes (1) embryonic precursors that differentiate into self-renewing tissue-resident macrophages and (2) bone marrow-derived haematopoietic stem cells (HSCs) that differentiate into blood monocytes 11. Specifically within the peritoneum, Cd11b + F4/80 + large peritoneal macrophages (LPMs) appear to be embryonically-derived, while Cd11c + MHCII + small peritoneal macrophages (SPMs) are source...

Background DNA variants in APOL1 associate with kidney disease, but the pathophysiologic mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNA-seq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types. Methods We performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-g, or the combination of IFN-g and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared with isogenic G0 controls. Results Both G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells. Organoids expressed detectable APOL1 only after exposure to IFN-g. scRNA-seq revealed cell type-specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids. Conclusions Single-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1-mediated kidney disease.